Complexes of phosphanilic acid and 9-amino-3-nitroacridine

ABSTRACT

Complexes of phosphanilic acid and an aminoacridine possess broad-spectrum antibacterial and antifungal activity when used topically. The preferred complex is a 1:1 complex of phosphanilic acid and 9-amino-3-nitroacridine.

United States Patent Pagano [54] COMPLEXES OF PHOSPHANILIC ACID AND 9-AMINO-3-NITROACRIDINE [72] Inventor: Joseph F. Pagano, Paoli, Pa.

[73] Assignee: Smith Kline & French Laboratories,

Philadelphia, Pa.

[22] Filed: March 10, 1970 [21] Appl. No.: 18,312

[52] US. Cl ..260/279 R, 260/502.5, 424/267 [51] Int. Cl. ..C07d 37/24 [58] Field of Search ..260/279, 502.5, 502.1

[56] References Cited UNITED STATES PATENTS 2,092,131 7/1937 Mietzsch ..260/279 [451 Sept. 26, 1972 Primary Examiner-Donald G. Daus Attorney-William H. Edgerton, Richard D. Foggio, Joan S. Keps, Alan D. Lourie and Joseph A. Marlino 5 7] ABSTRACT Complexes of phosphanilic acid and an aminoacridine possess broad-spectrum antibacterial and antifungal activity when used topically. The preferred complex is a 1:1 complex of phosphanilic acid and 9-amino-3- nitroacridine.

3 Claims, N0 Drawings COMPLEXES F PHOSPHANILIC ACID AND 9- AMINO-3-NITROACRIDINE This invention relates to compositions having antibacterial activity. In particular, the invention relates to complexes of phosphanilic acid and an acridine compound.

This invention, in its broadest aspect, comprises complexes of phosphanilicacid and an antibacterial acridine.

- The invention, in a more limited aspect, comprises complexes of phosphanilic acid and an aminoacridine, preferably a 9-amino, 3-arnino, or 6-aminoacridine,

' said acridine having antibacterial activity.

, The invention, in a still more limited aspect, comprises l:'l and 2:1 complexes of phosphanilic acid (I) and a 9-amino,3-amino, or 6-aminoacridine.(ll), said acridine being additionally substituted with one or more amino, methyl, ethyl, chloro, bromo, nitro, methoxy, ethoxy, cyano, phenyl, orrelated groups and also havingantibacterial activity. In formula II, each R is NH, orH, at least one of such groups being N11 and each R is' hydrogen, amino, methyl, ethyl, methoxy, ethoxy, chloro, bromo,nitro, cyano, or phenyl.

The invention, in its preferred aspect, comprises the 1:1 and 2:1 complexes of phosphanilic acid and 9- ;amino-B-nitroacridine (Ill).

, ing together pure samples of phosphanilic acid and the desired acridine. Complex formation is most concluding dimethylformamide; tetramethylene sulfone, or

hexamethyl phosphoramide, may be used. Choice of appropriate solvents is wellwithin the skill of theart.

activity is demonstrated for the preferred complexes of the invention, the 1:1 complex of phosphanilic acid and 9-amino-3-nitroacridine (SK&F-36387-2:1

complex of phosphanilic acid and 9-amino-3- nitroacridine .(SK&F-36387+-J,) in a number of test systems including the agar-inclusion method.

In this system,the test compound isincorporated in agar attwo-fold levels from 200 Lg/ml downward, and the test organisms inoculated onto-the hardened agar surface. Activity is measured as the lowest concentra tion which prevents growth of the test organism.

Results are shown in Table 1.

TABLE -1 IN VITRO ACTIVITY OF SK&F 36387-J AND SK&F 36387-1:

Average MIC pglnl Range) Organism SK&F 363874 SK8tF 36387-1,

- Sraphylocwcus aureus 15.6 26.1

(6 strains) (6.3 25) (12.5 50) Staphylococcus albus 8.2 8.6

(4 strains) (4.7 12.5) 6.3 12.5) Slreprococcusfaemlu (1 strain) 6.3 12.5 Streptococcus pyogencs 7.6 15.3

strain (0 8- 12.5) (1.6-25) Klebsiella pneumoniae 9.4 22.9

.(3 strains) (3.1 12.5) (6.3 37.5) Esrhelichia coli 6.3 7.5

(5 strains) (6.3) (6.3 9.4) Salmonella .rp. 14.6 25

(3 strains) (12.5- 18 8) (25) Shigella .tp.

(1 strain) 3.1 3.1 Candida albicans' 15.0 25.0 (5 strains) (12.5 25) (25) Pseudomonas .rp. 5.1 7.1

(21 strains) (16-125) (0.8-25) Fungi:

Fusarium oxysporum Penicillium cim'num 6.8 l 1.0

(SI-12.5) (31-25) Aspergillus niger Cryplococcus neoformans Blastomyce: dermatitwir Trichophyton mentagmphyle:

The 1:1 complex was further tested for activity against a total of 77 strains of Gram-positive and negative bacteria, including 40 strains of Pseudomonas, an organism found difficult to cope with clinically, plus 8 strains of yeast and fungi. Of this spectrum, almost all were recent clinical isolates.

Tested simultaneously with SK&F 36387-1 were two highly regarded commercial topical agents, hexachlorophene and nitrofurazone.

In addition to the above, a tube-dilution test was carried out against 6 strains of Proteus sp., a Gram-negative organism which frequently appears in burn cases and in mixed infections.

Results of these experiments are detailed in Tables 2,

The results show that both complexes possess broad spectrum activity against all strains tested and, in parj ticular, the 1:1 complex (SK&F36387-'J) was effec tive against 40 hospital strains of Pseudomonas sp. and various clinical strains of Proteus sp. In contrast, hex- I achlorophene showed poor activity against Gram-negative organisms and nitrofurazone was ineffective against all strains of Pseudomonas and fungi tested.

An antimicrobial agent, to be effective in vivo, must be capable of retaining its activity in the presence of a variety of substances, including whole blood, serum, sebum, pus, and other body fluids. In addition, a topical antimicrobial intended for human use must also remain effective when applied to skin which retains traces of soap.

In vitro experiments were carried out to evaluate the effectiveness of SI(&F 36387-J, hexachlorophene, and nitrofurazone against Staphylococcus aureus and Pseudomonas sp. in the presence of serum, soap, and milk. Minimum inhibitory concentrations (MIC) for each condition were established and compared to the MIC in normal medium without neutralizer. The figures presented in Table 7 are expressions of the ratio of MIC in neutralizer broth to MIC in normal broth.

TABLE 7 EFFECT OF NEUTRALIZING SUBSTANCES ON ANTIBACTERIAL ACTIVITY Effect of Neutralizer on MIC Test Compound Organism Soap Milk Serum Staph.

, aureus SK&F FDA 209. 1.4x 1.5x 2x 38387-1 Pseudomonas sp. mi 1879 11 2x 2x Staph. aureus Hexachloro- FDA 209 1X 4.5x 250 phene Pseudomonas sp. I-Il-l 1879 4X 64X 128x Staph. aureus Nitrofurazone FDA 209 0.5x 1X 2X Not Not Not Pseudomonas Active Active Active sp. HB 1879 at 1000 at 1000 at 1000 Lg/m1. Lg/ml. uglrnl.

*Expressed in multiples of MIC in normal broth (no neutralizer added).

The results show that SK&F 36387-1 is able to retain antimicrobial activity in the presence of neutralizing substances. In media containing 20 percent serum and 5 percent milk (representative of organic and lipid materials), loss of activity was very slight. In the presence of soap, the anti-Pseudornonas activity of SK &F 36387-J was reduced moderately, but its anti- Staphylococcus activity suffered little. Hexachlorophenes activity in the presence of soap diminished slightly. In the presence of milk, loss of antibacterial activity became greater, and in the presence of 20 percent serum, activity loss was very severe, with MICs ranging from 128X to greater than 250x the drug level found effective in normal medium. Nitrofurazone lost little of its activity against Staphylococcus in the presence of serum, milk, or soap. Against Pseudomonas, however, nitrofurazone displayed no activity at 1,000 ug/ml under any in vitro test conditions, including medium containing no neutralizing substances.

The substantivity of a compound, or its ability to be retained by the cells of the skin, is a valuable characteristic for topical agents. Hexachlorophenes ability to do so is well known and serves as one of its major attractions, offsetting its limited spectrum of activity. Experiments were carried out to assess the in vitro substantivity of SK&F 36387J, hexachlorophene and nitrofurazone using an in vitro method utilizing skin discs. In this system, standardized calf skin discs are immersed in solutions of drug for 30 minutes, then vigorously washed in running water for extended period of time. Washed discs are then placed on agar surfaces inoculated with Staphylococcus aureus and Pseudomonas sp. for 30 minutes, after which the discs are removed and the plates incubated. A substantive compound is defined as one which is sufficiently retained by the calf skin discs to produce zones of inhibition, despite the vigorous washing. The results of these in vitro experiments are shown in Table 8. Data are expressed as the diameter of the zones of inhibition produced by washed calf skin discs after exposure to solutions of the test compounds.

TABLE 8 SUBSTANTIVITY TEST ZONES OF INHIBITION (mm.) PRODUCED BY WASI-IED CALF SKIN Average of two tests SK&F 36387-J was found to be substantive at both 005 and 0.2 percent. Activity of the compound against Staphylococcus aureus and Pseudomonas sp. was still evident after wash cycles of up to 2% hours. I-Iexachlorophene also displayed substantivity at both high and low test levels after prolonged washing. Nitrofurazone remained substantive after maximum wash time only at its higher test level; substantivity at the lower test level was no longer evident when disc wash time was extended to 2% hours.

Antimicrobial agents can be shown to exert either of two types of efi'ects on bacterial cells: a truly lethal, irreversible, action termed bactericidal, or a reversible action in which the organism, rendered free of the agent, can again multiply, in which case it is termed bacteriostatic. To determine whether the activity of SK&F 36387-J is bactericidal or bacteriostatic, the following experiment was carried out. Two-fold dilutions of SK&F 36387-J in broth were inoculated with three strains of Gram-negative bacteria (Proteus) and two strains of Gram-positive bacteria (Staphylococcus). After 24 hours incubation the minimum inhibitory concentration (MIC) of SK&F 36387-J was observed. Incubation was continued for an additional 48 hours (total 72 hours) and the end points again observed. To determine if viable bacteria remained in tubes beyond the 72 hour end points, despite the absence of any such evidence, aliquots of each were transferred to complexfree medium. After incubation, tubes were examined for evidence of bacterial growth; if present, the action of SK&F 36387-J would then be known to be bacteriostatic; if absent, the drug action would be bactericidal. Results are shown in Table 9.

TABLE 9 EFFECTS OF PROLONGED INCUBATION ON SK&F 36387-J END POINTS Minimum Inhibitory Conc. (MIC) SK&F 36387-.I(p.g/m1.)

The results show that the MIC s changed only slightly with prolonged incubation. Sub-culturing of aliquots from tubes beyond the 72 hours MIC produce d no sign of viable organisms; the action of SK&F 36387-J against both Gram-positive and Gram-negative bacteria is seen to be bactericidal.

Data in Table 4 indicate the anti-Pseudomonas activity of SK&F 36387-J, compared with that of hexachlorophene and nitrofurazone. In further experiments, SK&F 36387-J was compared with compounds well-known for their anti-Pseudomonas properties and therefore used for treatment of burns. These compounds were gentamicin sulfate, mafenide hydrochloride, and silver sulfadiazine. These compounds are all reputed to be highly effective. Compounds were evaluated in an in vitro, agar-inclusion system for activity against 40 hospital strains of Pseudomonas sp. of these experiments are presented in Table 10.

TABLE 10 IN VITRO ACTIVITY AGAINST PSEUDOMONAS SP. (AGAR-INCLUSION METHOD) Minimum Inhibitory Conc. ag/m1) Pseudomonas sp. SK&F Gentamicin Silver Mafenide Strain 36387 (Base) sulfadiazine (he!) HH 29913 5.5 6.9 25 400 HH 29980 3.1 6.9 25 400 HH 29998 1.6 6.9 25 400 HH 28592 1.0 13.7 25 400 HH 29308 6.0 6.9 25 400 HH 28858-A 25 6.9 25 400 HH 28858-3 25 3.5 25 400 l-lH 28858-C 18.7 6.9 25 400 HH 29063 18.7 6.9 25 400 HH 29080 4.7 6.9 25 400 HH 29107 15.6 6.9 25 400 1111 22858 25 6.9 25 400 HH 29156 7.8 6.9 25 400 HH 29260 18.7 6.9 25 400 I'll-1 29310 12.5 6.9 25 400 HH 29322 2.7 6.9 25 400 HI-l29323 25 6.9 25 400 HH 29334 2.3 13.7 25 400 HH 29621 9.4 3.5 12.5 400 I-IH 29674 10.9 13.7 25 400 HH 29628 6.3 3.5 25 400 HR 29689 4.7 6.9 25 400 HH 29704 3.1 6.9 25 400 I-lI-I 29714 4.7 6.9 25 400 HH 22869 4.7 10.3 18.7 400 HH 29163-A 12.5 3.5 25 400 HH 29260 9.4 6.9 12.5 400 HH 29270 6.3 10.3 12.5 400 111-1 29790 2.3 6.9 12.5 400 HH 29830 4.7 3.5 l2..5 400 HH 29897 1.6 13.7 12.5 400 HH 22074 12.5 6.9 12.5 400 HH 22179 9.4 6.9 12.5 400 HI! 22167 6.3 13.7 12.5 400 HH 22168 18.7 6.9 400 l-lH 22978 3.1 6.9 25 400 HH 22869 6.2 6.9 25 400 HH 22979 3.1 6.9 18.7 400 RH 56 12.5 400 ATCC 19660 NIH 180 3.1 400 Average Pseudomonas MIC (40 strains) 9.4 7.5 21.7 395 Average of two or more tests The results show that SK&F 36387-J is highly active against all strains of Pseudomonas sp. tested. Of the other compounds, only the antibiotic gentamicin possesses the order of activity shown by SK&F 36387-J. Silver sulfadiazine was consistently effective at levels approximately 2X higher than SK&F 36387-J and gentamicin. Mafenide, however, displayed activity only at test levels 8X to 16X higher than the other compounds. An additional experiment was conducted to determine the ability of SK&F 36387-J to prolong the lifetimes of burned mice infected with Pseudomonas. Male, albino, Charles River mice (14-18 grams) were burned by immersing the tails of etherized animals in water at 70C for 5 seconds. Inoculation with Pseudomonas aeruginosa (ATCC No. 19660, NIH No. 180) was carried out 2 hours later by dipping the burned tails in an 18 hour broth culture of the organism. The tails were then encased in soft latex tubing three-sixteenths inch bore, one-sixteenth inch wall) which was stapled to the loose skin at the base of the tail with Michel wound clips. Five hours post-infection, a single applica- 60 tion of the test ointment (approximately 1.0 ml) was injected into the sheath enclosing the tail and the end of the sheath closed with a size 00 cork to avoid ointment loss. Mice were observed daily for deaths during the entire 12 day duration of the test. Heart blood and the 5 kidneys of mice which died were cultured for viable organisms; in all cases, Pseudomonas was recovered, indicating a Pseudomonas septicemia. Results are shown in Table l 1.

TABLE II -7 7 Y Topical chemotherapy of Pseudomonas septicemia in burned mice Concentratoin N of Percent survivors Days post-infection us o Ointment type 1 percent mice 1 2 3 4 5 6 7 8 9 10 11 12 flKiQF 86387-3. l 0. 2 100 100 97 97 97 97 98 93 93 90 Placebm. 70 100 100 88 59 51 46 34 28 25 21 3 1 Nltrolurezonc 0.2 20 100 100 95 80 65 60 55 65 45 35 30 30 30 (luntumlcin... 0.12 30 100 100 100 90 83 83 80 73 67 63 60 60 60 None(lnfcetion eontrol) 120 100 100 54 23 16 15 l3 l2 9 9 9 8 8 lium control... 40 100 100 100 100 98 91 90 90 90 90 90 90 90 1 Average of 3 tests. 2 Average of 2 tests.

The results indicate that SK&F 36387-1, in a 0.2 percent ointment, provided excellent protection against the. Pseudomonas aeruginosa infection produced in burned mice. The protection provided by a-single application waslong lasting, with 90 percent of the animals still protected on the l2th'day after treatment. 'Gentamicin ointment provided initial protection equal to that ,of SK&F 36387-4, ,but its protective effect decreased with time. Commercial nitrofurazone ointment (0.2 percent) was protective for the firstfew days of the test only; by the tests end, 70 percent of the treated animals had died. Untreated, infected animals quickly succumbed to their Pseudomonas infections! (77 percent died by day 3); the burn control animals (no infection) survived the test with only 4/40 deaths.

The-complexes of the present invention are intended? to be used topically.- They are forrnulatedfor such use 5 by combining-them with appropriate topically acceptable carriers or vehicles and other materials to form antiseptic solutions, soaps, shampoos, lotions, ointment's, creams, toothpaste, powders, mouthwashes, etc. Those skilled in the art-of pharmaceutical chemistry will be able to formulate these compositions without extensive experimentation utilizing normal skills as well as themformation provided in the following examples. A solution should contain about 0.0l-l0 percent of the complex. A topical ointment should contain about 0.025-0.5 percent of the complex.

The following examples are intended to illustrate the complexes and compositions of the present invention, but are not to be constructed as limiting the scope thereof; a I I EXAMPLE 1 1:1 Complex of Phosphanilic Acid and 3-Nitro-9- aminoacridine To a warm solution of 180 g. (0.75 m.) of 3-nitro-9- aminoacridine in 1800 ml. of dimethyl sulfoxide was added a hot solution of 146 g. (0.84 m.) of phosphanilic acid in 1,800 ml. of dimethyl sulfoxide. The dark red solution was stirred vigorously while 3,600 ml. of hot water was rapidly added. The reaction mixture was cooled to about 10C. and the product separated by filtration, washed with water and dried invacuo at 100C. to give 296 g. (95.5 percent yield) of 3-nitro-9- aminoacridine phosphanilate.

EXAMPLE 2 2:1 Complex of Phosphanilic Acid and 3-Nitro-9- aminoacridine To a warm solution of 42 g. (0.176 m.) of 3-nitro-9- aminoacridine in 450 ml. of dimethyl sulfoxide was added a hot solution of 68.5 g. (0.39 m. 'ofphosflanilsolution was stirred vigorously while 5,600 ml. of hot water was rapidly added. The reaction mixture was cooled to about 10C. and the product separated by filtration, washed with water and dried in vacuo at 100C.

to give 98 g. percent yield) of 2-nitro-9- aminoacridine, diphosphanilic acid complex.

EXAMPLE 3 In the same manner as in Example 1, a slight excess of phosphanilic acid may be reacted with each of the following acridines to 'give the corresponding l:l complexes.

9-arninoacridine 3-aminoacridine 3,9-diaminoacridine 3,6-diaminoacridine 4,9-dia'minoacridine 3,7-diaminoacridine 9-amino-4-methylacridine 9-amino l-methylacridine 9-amino-3-methylacridine 9-amino-2-methylacridine 9-amino-3-chloroacridine 9-amino-2-chloroacridine 9-amino l -chloroacridine 9-amino-4 chloroacridine 3-amino-6-chloroacridine 3-amino-7-chloroacridine 1,6-diaminoacridine 2,6-diaminoacridine l ,9-diaminoacridine 2,9-diaminoacridine 9-amino-2,4-dimethylacridine 9-amino-4,5-dimethylacridine 9-amino-4-ethylacridine 9-amino- 1 -methoxyacridine 9-amino-2-methoxyacridine 9-amino-3-methoxyacridine 9-amino-4-methoxyacridine 9-aminol -.chloroacridine 9-amino-2-chloroacridine 9-amino-3-chloroacridine v9-amino-4-c hloroacridine 9-amino-1 -nitroacridine .9-amino-2-nitroacridine 9-amino-3-nitroacridine 9-amino-4-nitroacridine 9-amino-2-phenylacridine 9-amino-4-phenylacridine 9-amino-2-cyanoacridine 9-methylaminoacridine 9-amino-2-carbomethoxyacridine 9-amino-2-carbamoylacridine In the same manner as in Example 2, a slight excess over 2 equivalents of phosphanilic acid may be reacted with 1 equivalent of each of the acridines named in Example 3 to give the corresponding 2:1 phosphanilic acid-acridine complexes.

EXAMPLE 5 Examples of suitable formulations of the complexes are as follows:

Antiseptic solution 0.05% w/v solution of the 1: 1 complex of phosphanilic acid and 9-amino-3-nitroacridine in 50 percent glycerin. Pure glycerin may also be used as solvent. Topical ointment (water-washable, oil-in-water emulsion) 1:] complex of phosphanilic acid and 0.200

9-amino-3-'nitroacridine Light liquid petrolatum 5.000 Cetyl alcohol 4.000

Stearyl alcohol 6.000

Tween 60 (Polyoxyethylene sorbitan 2.100

monostearate) Span 60 (Sorbitan monostearate) 2.100

Propylparaben 0.150

Ethylparaben 0.100

Glycerin 10.000

Water A 70350 Topical ointment 1:1 complex of phosphanilic acid an 0.20

9-amino-3-nitroacridine Other complexes from Examples 2, 3 and 4 may similarly be formulated into solutions, ointments, and suspensions.

I claim:

1. A 1:1 or 2:1 complex of phosphanilic acid and 9- amino-3-nitroacridine.

2. A complex according to claim 1, consisting of 9- aminQS-nitroacridine and 2 equivalents of phosphaniic acid.

3. A complex according to claim 1, consisting of 9- amino-3-nitroacridine and 1 equivalent of phosphanilic acid. 

2. A complex according to claim 1, consisting of 9-amino-3-nitroacridine and 2 equivalents of phosphaniic acid.
 3. A complex according to claim 1, consisting of 9-amino-3-nitroacridine and 1 equivalent of phosphanilic acid. 